Stimulating microbial growth



Patented Dec. 20, 1966 3,293,145 STIMULATIN G MICROBIAL GROWTH Richard I. Leavitt, Pennington, and Israel J. Heilweil, Princeton, N.J., assignors to Mobil Oil Corporation, a corporation of New York No Drawing. Filed Aug. 26, 1964, Ser. No. 392,319 5 Claims. (Cl. 19580) This invention relates to a method for stimulating the growth of microorganisms while supplying to them a hydrocarbon as the sole source of carbon for both growth and energy.

Increasing the growth rate of microorganisms is of importance in view of the ability of many of them to synthesize useful products such as proteins, amino acids, vitamins, lipids, polymers, and other compounds of value. These products may be formed in the cells or secreted by them into the medium in which the microbe is growing, and in either case, are recoverable. Their rate of formation increases with the rate of cell growth, and as a result, less time is required before harvesting of the cells may take place. In some cases, the invention provides greater yields of cells than are otherwise attainable. The invention is also of importance to the microbial oxidation of hydrocarbons. Thus, certain hydrocarbons, such as n-decane, are not only capable of being utilized by a microorganism as the carbon source therefor but also, and during the course of being utilized, are converted by the microorganism into various oxygenatedhydrocarbon products, including aldehydes, ketones, acids, and esters, many of which are of higher value than the starting hydrocarbon. A frequent difficulty in microbial oxidations is the relatively slow rate of growth of the microbe. By aid of this invention, this difficulty may be considerably lessened.

The invention, briefly, comprises incubating a microorganism as herein defined with an aqueous mineral salts medium in the presence of oxygen and, as sole source of carbon, an aliphatic hydrocarbon and, further, in the presence of a small amount of a nonionic surface active agent which acts as a growth stimulator.

The preferred microorganism is an Achromobacter, as illustrated by species such as A. xerosis, A. gutatws, A. superficialis, A. parvulus, and A. cycloclastes. Also useful is the genus Nocardia, particularly N. salmonicolor, but also including N. asteroides, N. opaca, N. corallz'na, and N. rubra.

The aliphatic hydrocarbon which forms the sole source of carbon for the microorganism is a saturated or unsaturated, straight or branched chain hydrocarbon having up to to 30 or more carbon atoms. Saturated straight chain alkanes having up to 20 carbons are preferred, particularly those which are liquid at the temperatures employed. The amount of hydrocarbon is that required to provide carbon for growth and energy and may be chosen for any desired growth period, although it may also be added from time to time to a culture mixture as may be necessary. It is desirable to add only the required amount so as to avoid separation problems at the end of the growth period.

The nonionic surface active agent is preferably a compound having an aromatic nucleus, such as a phenyl ring, substituted by a side chain of hydrophilic character such as a polyoxyethylene group, and by a side chain of lipophilic character such as an alkyl group. Agents of this kind are frequently referred to as polyoxyethylene alkyl aryl ethers, obtainable as by reacting an alkylphenol with ethylene oxide. The agent is useful in concentrations as low as 0.001% by weight of the aqueous mixture which is incubated, but usually is 0.01 to 0.1% of said mixture and may range up to 0.5, l, or 5% or more. Generally,

the lower concentrations are preferred. It will be noted that the agent is a non-ionizing compound. Ionizable agents are unsuitable, it having been found that they do not stimulate cell growth.

Other useful nonionic agents are polyoxyethylene glycols and alkyl ether derivatives thereof; and methoxy polyoxyethylene glycols and their ester derivatives.

Still other agents are fatty acid esters, including monoand diesters, formed from a polyol and a fatty acid. The polyol may be glycol, glycerol, sorbitol, sorbitan, mannitol, propylene glycol, polyoxyethylene glycol, etc., and the acid may be an aliphatic monocarboxylic acid, saturated or unsaturated, straight or branched chain, preferably having from 12 to 18 carbon atoms. Examples are glycerol monoand dilaura-tes, glycerol monoand dioleates, glycerol monoand distearates, glycerol monopalmitate, glycerol monomyristate, propylene glycol monostearate, propylene glycol monopalmitate, propylene glycol monooleate, and mixtures thereof. Also, sorbitan laurate, sorbitan monoand tristearates, sorbitan monoand trioleates; mannitan stearates, palmitates, and laurates; mono-, di-, and triglycerides of fatty acids like oleic, palmitic, and stearic; glycerol sorbitan laurate, also polyoxyethylene laurates, stearates, oleates, and palmitates; and polyoxyethylene sorbitan palmitates, oleates, stearates and laurates. Sucrose monoand dipalmitates are suitable, as well as other monoand diesters of sucrose and fatty acids of, preferably, at least 12 carbon atoms, including sucrose monolaurate, sucrose monostearate, sucrose monooleate, sucrose dilaurate, sucrose dimyristate, sucrose distearate, sucrose dioleate, and the like.

Other suitable nonionic agents are fatty acid derivatives formed by reaction of a fatty acid and ethylene oxide. Also alcohol derivatives formed by reaction of a fatty alcohol (having at least 8 carbons) with ethylene oxide. Still other agents are fatty amide derivatives having an oxygenated side chain of hydrophilic character, with the lipophilic portion of the compound being due to the amide grouping. These derivatives may be formed by reaction of a fatty acid amide and ethylene oxide, or by reaction of a fatty acid or ester with an alkanolamine.

The mineral nutrient comprises mineral salts which furnish ions of ammonium, nitrate or nitrite, potassium, ferrous or ferric, calcium, magnesium, phosphate, sulfate, as well as ions of trace elements such as zinc, manganese, copper, and molybdenum. Water is included in the nutrient mixture, so that most of these mineral salts will usually be present in sufficient quantity in ordinary potable water supplies. However, it is desirable to add the salts to the mixture to insure their presence in sufficient quantity for growth. Usually the mixture consists primarily of water, which may constitute up to 99%, or

more, by weight of the liquid phase of the mixture, although it may also constitute a lesser portion, going down to 50% by weight of the liquid phase. Generally, any proportion of water heretofore employed in microbial synthesis may be used.

A water-soluble nitrogen compound should be present in the mineral salts solution and preferably this compound is urea because it appears that a condition of synergism exists when urea and the nonionic agent are present together in the culture, i.e., the growth of the cells is greater when these two compounds are present together than when one of them is absent. Further, the synergism appears to be more marked when, in addition to urea and the nonionic agent, there is also present a water-soluble inorganic ammonium salt such as ammonium sulfate. Experiments illustrating these procedures are set forth in Example 3. The amount of urea is suitably about 0.15% by weight of the aqueous mineral salts solution but may range from 0.015 to 1.5%, and may be used in even higher concentrations going up to or or more. Similarly, the inorganic ammonium salt may suitably be used in a concentration of about 0.1% but may range from 0.01 to 1% and higher, say to 5 or 10%.

A suitable mineral salts medium may be listed as follows, the components being dissolved in enough water to make one liter of solution:

The method comprises incubating the microorganism in the mineral nutrient, in which the hydrocarbon and nonionic agent are present, with stirring, and, after growth is obtained, separating the cells from the medium. Recovery of the desired components from the cells or from the supernatent may then be carried out. The nonionic agent is unchanged and may be recovered. In some cases separation of the cells may not be necessary, as Where the entire incubated mixture is used in or as an animal feed or as a fertilizer material.

During incubation, which can be done in conventional reactors, the culture mixture is maintained under conditions to insure optimum growth of the microorganism. The temperature for example should be maintained between about 20 and about 55 C., preferably from 20 to 30 C. The pH is maintained near neutrality, namely, about 7.0, although it may range between about 5.5 and 8.5. It is desirable to maintain the mixture in a condition of agitation as by shaking, or by using propellers, paddles, rockers, stirrers, or other means ordinarily employed for effecting agitation of a liquid mixture. Suitably, the reactors are open to the atmosphere, and with agitation of the mixture, the surface thereof exposed to the atmosphere is continuously renewed and oxygen is taken up. However, where the hydrocarbon is normally gaseous, the reactors are closed, and oxygen may be supplied by bubbling it or air through the mixture, preferably in company with the hydrocarbon, thereby also providing desired agitation.

Microbial syntheses conducted by the present method may be completed in times as short as one or two days. The incubation period may of course extend longer, but it is of interest to note that many microbial conversions, including syntheses, have in the past required periods of a week or two, or more, within which to produce appreciable growth. tion, the time may be reduced to less than a day, if desired, as by starting out with a quantity of cells previously grown and adding to them a nonionic surface active agent of the kind described. In this way, the yield of cells may be doubled within a space of time corresponding to their generation time, which may run as low as 3 or 4 hours. Use of pregrown cells in the foregoing manner may also be advantageous in other ways, as inwalls and extracting the products from the resulting debris,

and thereafter separating the extract further as desired. Extracellular products are recoverable by conventional methods.

. Substantial increases in rate of cell growth and in In some cases, according to the inven- A soil-isolated organism identified as a member of the genus Achromobacter was inoculated into several 250 ml. Erlenmeyer flasks, each containing 50.0 ml. of the mineral salts medium whose qualitative and quantitative composition is listed above and to which 0.3 ml. decane had been added. The decane functioned as the sole carbon source. The following protocol describes various additions which were made to the flasks:

No. l--None.

No. 2-Renex 688, a nonionic surface active agent of the formula p-C H -C H -O(CH CH O) CH OH. Final concentration 0.01%.

No. 3Renex 688. Final concentration 0.001%. The flasks were incubated at 25 C. with shaking and the optical density of the various cultures determined at periodic intervals. Data obtained are as follows:

Optical Density Agent Flash No. Conan,

percent At 24 At 48 At 72 Hrs. Hrs. Hrs.

After 38 hours decane could still be detected in flasks Nos. 1 and 3 but the decane in flask No. 2 was exhausted. Additional decane (0.5 ml.) was added to each flask at this time and the flasks reincubated an additional 24 hours. As may be seen, the addition of nonionic agent resulted in a Significant increase in the growth rate of the culture.

Thus, after 48 hours of incubation, flask No. 2 exhibited in place of the Renex 688, the experiment being otherwise the same, there was no stimulation of cell growth. And in a further experiment using a cationic surface active agent of the formula C H -C H -NBr in place of the Renex 688, there was no stimulation of cell growth.

Optical density was measured by testing samples of each culture mixture for the adsorption of visible light rays of a wave' length of 400 millimicrons (0.4 micron) in a Bausch and Lomb colorimeter. The resulting data are expressed above as optical density, and the relation between optical density and cell growth is as follows: an optical density of 1.0 is equivalent to approximately 2.1 g./l. of cells, dry weight.

Example 2 Cell growth was carried out on a larger scale using a bacterium identified as Nocardia salmonicolor 107-332. It was grown in two S-liter fermenter vessels, each containing 3 liters of mineral salts medium as used in Example 1. One of the vessels contained Renex 688 at a concentration of 0.001% and the other vessel contained no agent, serving a a control. During the entire incubation period n-butane and air were bubbled through the culture liquid in a ratio of 10 parts air to one part butane. The vessels were incubated with agitation at 30 C. and growth in each fermenter determined in terms of optical density increase at regular intervals. Both cultures grew at approximately the same rate during the first nine days of incubation. However, after this period, the culture without added agent stopped growing and exhibited a decrease in density; it therefore was harvested. At this point, the cells of the first culture continued to grow and divide but at a lower rate than before; they continued to increase in density for a total of 21 days at which time they too were harvested. A comparison of the dry weights of the two cultures showed that the addition of the small amount of agent resulted in an increase in cell yield of approximately 40%.

Untreated Cells, Treated Cells,

g./liter g./liter The effect of urea on the microorganism of Example 1 was observed by preparing three flasks, each containing 50 ml. of the mineral salts medium of Example 1, to which 0.4 ml. of decane had been added. Each flask contained a water-soluble source of nitrogen, flask No. 1 having 1.0 g./l. of ammonium sulfate, flask No. 2 having 1.0 g./l. ammonium sulfate plus 1.5 -g./l. urea, and flask No. 3 having 1.5 g./l. urea. Runs were made in which the surface active agent was omitted and also in which the agent was added, in each case the agent being the same as in Example 1. The culture mixtures were all incubated at 25 C. with shaking for a period of 72 hours after which the following optical density data were obtained.

As may be seen, the effect on cell growth was most marked in the culture which contained both urea and ammonium sulfate and to which the surface active agent had been added, as in No. 2. When urea was excluded, as in No. 1, the effectiveness of the agent was very greatly diminished. Control runs in which either decane or the cells were omitted showed no growth. It was further ascertained that the effect of the urea was not due to possible elevation of the pH.

A convenient source of supply of urea and aliphatic hydrocarbons for the process is the so-called urea-normal paraffin adduct which is obtainable in the dewaxing of lubricating oils. In the latter operation, a lube oil having an elevated pour point is dewaxed by extraction with a saturated aqueous solution of urea, the normal paraffins in the oil forming a solid adduct with the urea, and this adduct is separable from the mixture, leaving a dewaxed lube oil of reduced pour point. The adduct is a crystalline material, often termed an inclusion complex, and can also be formed by mixing urea with a normal alkane having at least 6 carbons. About 6 moles of urea per mole of n-alkane are required when the latter has 7 carbons, and about 21 moles of urea per mole of n-alkane when the latter contains about 28 carbons. The

alkane fills the interstices normally present in urea crystals to form the complex, and on placing the complex in water, the urea dissolves, releasing the alkane.

The adduct or complex from any source, but particularly from a dewaxing operation because of its low cost, may be introduced to the culture to provide the necessary urea and aliphatic hydrocarbon. Of interest is the fact that at the conclusion of the growth period, the entire culture mixture may be taken and utilized for cattle feed; or if desired, the cells may be separated from the aqueous supernatent and then used as feed. Any excess urea associated with the cells need not be separated therefrom in view of the fact that urea is of value as an ingredient in prepared cattle feeds. A desirable procedure in this connection, is to grow the cells until all of the hydrocarbon introduced to the culture mixture with the complex is used up; in this way separation of any unused hydrocarbon is avoided in the event that the cells are isolated, and further, presence of hydrocarbon in the resulting cattle feed is avoided.

It will be understood that the invention is capable of obvious variations without departing from its scope.

In the light of the foregoing description, the following is claimed.

We claim:

1. Method of stimulating the growth of a microorganism selected from the group consisting of Achromobacter and Nocardia on an aliphatic hydrocarbon as the sole source of carbon for energy and growth which comprises incubating said microorganism in an aqueous mineral salt solution in the presence of oxygen and said hydrocarbon to form an incubation mixture, said solution containing 0.015 to 10% by weight of urea and 0.01 to 10% by weight of a water soluble inorganic ammonium salt, said mixture containing 0.001 to 5% by weight of a nonionic surface active agent, the joint use of said agent, urea, and ammonium salt having a synergistic effect on the growth rate of said microorganism as demonstrated by the growth rate is substantially greater than if a like incubation mixture were used but (1) which omits said agent, or (2) which omit said urea, or (3) which omits said ammonium salt, or (4) which omits said agent and urea, or (5) which omits said agent and ammonium salt.

2. The method of claim 1 wherein said hydrocarbon is a normal alkane having at least 6 carbon atoms, and wherein the hydrocarbon and the urea are added to the said incubation mixture in the form of .a solid crystalline water-decomposable urea-alkane inclusion complex.

3. The method of claim 1 wherein said microorganism is incubated in said mixture after having first been grown on said hydrocarbon and a conventional mineral salts nutrient to the maximum stationary phase.

4. The method of claim 1 wherein the microorganism is an Achromobacter.

5. The method of claim 1 wherein the microorganism is a Nocardia.

References Cited by the Examiner UNITED STATES PATENTS 2,697,061 12/1954 Harris et :al 19534 2,890,989 6/1959 Anderson l28 3,025,221 3/1962 Ciegler et al. 28 3,057,784 10/1962 Davis et al. 19528 3,169,099 2/1965 Davis 19534 3,201,327 8/1965 Beck 195-28 OTHER REFERENCES Beerstecher, Petroleum Microbiology, Elsevier Press Inc., New York, 1954, page 168.

Zobell, Advances in Enzymology, vol. 10, pages 443- 449.

A. LOUIS MONACELL, Primary Examiner. ALVIN E. TANENHOLTZ, Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent Non 3,293,145 December 20, 1966 Richard 16 Leavitt et a1.

It is hereby certified that error appears in the above numbered patant requiring correction and that the said Letters Patent should read as corrected below.

Column 5, line 2 8, for "of urea on the microorganism" read of urea on the growth of the microorganism column 6, line 39, for "the growth rate" read the fact that the growth rate Signed and sealed this 12th day of September 1967.

(SEAL) Attest:

ERNEST W. SWIDER Attesting Officer EDWARD J. BRENNER Commissioner of Patents 

1. METHOD OF STIMULATING THE GROWTH OF A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF ACHROMOBACTER SOURCE OF CARBON FOR ENERGY AND GROWTH WHICH COMPRISES INCUBATING SAID MICROORGANISM IN AN AQUEOUS MINERAL SALT SOLUTION IN THE PRESENCE OF OXYGEN AND SAID HYDROCARBON TO FORM AN INCUBATION MIXTURE, SAID SOLUTION CONTAINING 0.015 TO 10% BY WEIGHT OF UREA AND 0.01 TO 10% BY WEIGHT OF A WATER SOLUBLE INORGANIC AMMONIUM SALT, SAID MIXTURE CONTAINING 0.001 TO 5% BY WEIGHT OF A NONIONIC SURFACE ACTIVE AGENT, THE JOINT USE OF SAID AGENT, UREA, AND AMMONIUM SALT HAVING A SYNERGISTIC EFFECT ON THE GROWTH RATE OF SAID MICROORGANISM AS DEMONSTRATED BY THE GROWTH RATE IS SUBSTANTIALLY GREATER THAN IF A LIKE INCUBATION MIXTURE WHERE USED BUT (1) WHICH OMITS SAID AGENT, OR (2) WHICH OMITS SAID UREA, OR (3) WHICH OMITS SAID AMMONIUM SALT, OR (4) WHICH OMITS SAID AGENT AND UREA, OR (5) WHICH OMITS SAID AGENTS AND AMMONIUM SALT. 